mouse monoclonal ifnar2 neutralizing antibodies  (PBL Assay)

Journal: Cell Death & Disease

Article Title: Tp53 determines the spatial dynamics of M1/M2 tumor-associated macrophages and M1-driven tumoricidal effects

doi: 10.1038/s41419-025-07346-0

Figure Lengend Snippet: A Cytokine profiling of M0 and M1 CM. High-glucose RPMI supplied with 10% FBS is served as the medium control. B Effect of IFN-β, IFN-γ or combination treatments on p53 expression in A549 cells. Cells were treated with 1 ng/ml IFN-γ and/or 5, 10, and 20 ng/ml IFN-β for 3 days. The lower panel shows p53 protein levels normalized to β-actin, quantified by ImageJ. C IFN-β levels in M0 and M1 CM measured by ELISA. High-glucose RPMI supplied with 10% FBS is served as the medium control. D Synergistic effect of IFN-β and IFN-γ on apoptosis in A549 cells. Cells were treated with IFNs at indicated concentrations for 3 days. E Inhibition of M1-induced apoptosis through IFN-β neutralization. The combination treatment included 2 μg/ml each of IFN-β and IFN-γ neutralizing antibodies (nAb) and 4 μg/ml IgG as the negative control. F Effect of JAK inhibitor I on M1-induced apoptosis in A549 cells. DMSO served as the negative control. G Effect of JAK1, JAK2, and TYK2 silencing on M1-induced apoptosis in A549 cells. NC: siRNA negative control. H Effect of IFNAR1 and IFNAR2 silencing on M1-induced apoptosis in A549 cells. NC: siRNA negative control. I Inhibition of M1-induced apoptosis through IFNAR1 and IFNAR2 neutralization. M1 CM was supplied with neutralizing antibodies or 2 μg/ml IgG. Apoptotic cells were quantified using Annexin V staining and assessed by flow cytometry. Data are representative of at least two independent experiments and represented as mean ± SD. P -value for multiple group comparisons determined by two-way ANOVA, otherwise using one-way ANOVA and followed by Tukey’s post hoc test. ns not significant, P -value > 0.05; * P -value < 0.05; ** P -value < 0.01; *** P -value < 0.001. See Supplementary Fig. for gating strategy of apoptosis assay.

Article Snippet: For the receptor neutralization, 5 × 10 4 A549 cells were seeded in 24-well plates and pre-treated with goat polyclonal IFNAR1 (Abcam, Cambridge, UK) and mouse monoclonal IFNAR2 neutralizing antibodies (PBL Assay Science, Piscataway, NJ) for 4 h, followed by treatment with M1 CM for 3 days.

Techniques: Control, Expressing, Enzyme-linked Immunosorbent Assay, Inhibition, Neutralization, Negative Control, Staining, Flow Cytometry, Apoptosis Assay

Journal: Journal of Virology

Article Title: Engineered Oncolytic Poliovirus PVSRIPO Subverts MDA5-Dependent Innate Immune Responses in Cancer Cells

doi: 10.1128/JVI.00879-18

Figure Lengend Snippet: TLR3 activation in MDA5-depleted DM440 cells inhibits EMCV but not PVSRIPO. MDA5-depleted DM440 cells were pretreated with 1 μg/ml isotype-matched control antibody (mouse IgG2A) or IFNAR2 blocking monoclonal antibody (12 h), treated with 25 μg/ml extracellular poly(I·C) or vehicle (6 h), and then mock (0 h) or virus (MOI of 0.1; 18 h) infected. (A, B, and C) Lysates were collected from mock-, PVSRIPO-, and EMCV-infected DM440 cells and analyzed by immunoblotting for markers of viral translation (P2, 2BC, and 2C for PVSRIPO; 3Dpol for EMCV) and innate response [p-STAT1 (Y701), STAT1, and IFIT1]. Immunoblots are representative of two independent experiments. Bottom panels show the fold increase in IFIT1 after treatments (means ± SEM; n = 2). *, P < 0.05. (D) Viral protein expression in treated DM440 cells after PVSRIPO (2C) or EMCV (3Dpol) infection as a percentage of maximum detected viral protein (means ± SEM; n = 2). *, P < 0.05, for all 3Dpol differences. Differences in results for 2C were not statistically significant.

Article Snippet: For blocking IFNAR, either 2 × 10 4 or 4 × 10 5 cells were plated in 24-well tissue culture dishes in 0.5 ml of growth medium or in six-well tissue culture dishes in 1 ml of growth medium, respectively, containing 1 μg/ml mouse anti-human IFNAR2 antibody (clone MMHAR-2; PBL Assay Science), 1 μg/ml mouse IgG2A (R&D Systems) isotype control, or no antibody ( 43 ).

Techniques: Activation Assay, Control, Blocking Assay, Virus, Infection, Western Blot, Expressing

Journal: Journal of Virology

Article Title: Engineered Oncolytic Poliovirus PVSRIPO Subverts MDA5-Dependent Innate Immune Responses in Cancer Cells

doi: 10.1128/JVI.00879-18

Figure Lengend Snippet: Inhibition of p-STAT1 (Y701) in PVSRIPO or EMCV-infected DM440 cells by IFNAR blockade has no effect on either virus. DM440 cells were mock treated (no antibody; lanes 1 and 2) or pretreated with isotype-matched control antibody (lanes 3 and 4) or 1 μg/ml IFNAR2 blocking antibody (lanes 5 and 6) (24 h) and then cotreated with antibody and mock, PVSRIPO, or EMCV infected. (A and B) Lysates from cells mock and PVSRIPO infected or mock and EMCV infected at an MOI of 0.1 were collected at the indicated time postinfection and analyzed by immunoblotting as described in the legend of Fig. 1. Immunoblots are representative of two independent experiments. (C) Virus yields of PVSRIPO/EMCV-infected DM440 cells (MOI of 0.01) were determined at 18 hpi (means ± SEM; n = 3). No statistically significant differences were found.

Article Snippet: For blocking IFNAR, either 2 × 10 4 or 4 × 10 5 cells were plated in 24-well tissue culture dishes in 0.5 ml of growth medium or in six-well tissue culture dishes in 1 ml of growth medium, respectively, containing 1 μg/ml mouse anti-human IFNAR2 antibody (clone MMHAR-2; PBL Assay Science), 1 μg/ml mouse IgG2A (R&D Systems) isotype control, or no antibody ( 43 ).

Techniques: Inhibition, Infection, Virus, Control, Blocking Assay, Western Blot

Journal: PLoS ONE

Article Title: LPS Regulates SOCS2 Transcription in a Type I Interferon Dependent Autocrine-Paracrine Loop

doi: 10.1371/journal.pone.0030166

Figure Lengend Snippet: moDCs were exposed to LPS (A) for indicated time periods for mRNA measurement of IFNα, IFNβ, IFNγ and IFNλ1 or incubated with IFNα (Bi), IFNβ (Bii), IFNγ (Biii) and IFNλ1 (Biv) for indicated time periods for SOCS2 mRNA measurement by qRT-PCR. (C) moDCs were pretreated for 30 min with or without 30 ug/ml neutralizing anti-IFNR2 antibodies or IgG 2a isotype control mAb and then incubated for 4 h with or without LPS for SOCS2 mRNA measurement by qRT-PCR. Data shown are representative of three independent experiments and expressed as the –fold induction of the gene of interest at different time points compared to time point 0 where the values were set to 1(A–B) or different treatment conditions compared with medium that values were set to 1. Statistical significance of results with anti-IFNAR2 mAb compared with control mAb is indicated (C) (* p<0.05; ** p<0.01).

Article Snippet: Mouse anti-human IFNAR2 neutralizing antibodies (CD118; PBL Biomedical Laboratories, NJ, USA) and mouse IgG2a isotype controls (R&D systems, USA) were used at the concentration of 30 μg/ml.

Techniques: Incubation, Quantitative RT-PCR, Control